Neuroblastoma RAS viral oncogene homolog (N-RAS) deficiency aggravates liver injury and fibrosis

Progressive hepatic damage and fibrosis are major features of chronic liver diseases of different etiology, yet the underlying molecular mechanisms remain to be fully defined. N-RAS, a member of the RAS family of small guanine nucleotide-binding proteins also encompassing the highly homologous H-RAS and K-RAS isoforms, was previously reported to modulate cell death and renal fibrosis; however, its role in liver damage and fibrogenesis remains unknown. Here, we approached this question by using N-RAS deficient (N-RAS−/−) mice and two experimental models of liver injury and fibrosis, namely carbon tetrachloride (CCl4) intoxication and bile duct ligation (BDL). In wild-type (N-RAS+/+) mice both hepatotoxic procedures augmented N-RAS expression in the liver. Compared to N-RAS+/+ counterparts, N-RAS−/− mice subjected to either CCl4 or BDL showed exacerbated liver injury and fibrosis, which was associated with enhanced hepatic stellate cell (HSC) activation and leukocyte infiltration in the damaged liver. At the molecular level, after CCl4 or BDL, N-RAS−/− livers exhibited augmented expression of necroptotic death markers along with JNK1/2 hyperactivation. In line with this, N-RAS ablation in a human hepatocytic cell line resulted in enhanced activation of JNK and necroptosis mediators in response to cell death stimuli. Of note, loss of hepatic N-RAS expression was characteristic of chronic liver disease patients with fibrosis. Collectively, our study unveils a novel role for N-RAS as a negative controller of the progression of liver injury and fibrogenesis, by critically downregulating signaling pathways leading to hepatocyte necroptosis. Furthermore, it suggests that N-RAS may be of potential clinical value as prognostic biomarker of progressive fibrotic liver damage, or as a novel therapeutic target for the treatment of chronic liver disease.

mounted onto the silicone tube of a peristaltic pump. The peristaltic pump was calibrated with DPBS to obtain a laminar flow rate of approximately of 5.0ml/min to perfuse the liver. Next, the silicone tube was equilibrated with solution A and all air bubbles released. In the next step, the mouse was sacrificed by an overdose of isoflurane anesthesia and the abdomen was carefully opened along the linea alba. The intestines were moved out of the abdominal cavity to the right side of the animal in order to expose the vena cava inferior. Carefully, the catheter (20G or 26G) was inserted into the vena cava inferior and after proof of proper catheter placement the perfusion was started. After initiating the perfusion, the portal vein was immediately opened to allow a constant flow rate through the liver and subsequently a small piece of the liver was cut off for further analysis. A successful flushing of the liver is indicated by a loss of color of the liver. The liver was perfused with each solution (A,B and C), separately for 5 min or 25 ml. After the final solution C, the perfusion was stopped and the liver was dissected and transferred into solution D and incubated for 20 min at 37°C. To assure a proper digestion of the liver, solution D was inverted several times during the incubation time.
Next, solution D containing the digested liver was filtered through a 70 µm cell strainer and the filtered solution was centrifuged at 50x g for 1 min at 4°C. The pellet containing hepatocytes was kept for further analysis and the supernatant was transferred to a new falcon and subsequently centrifuged at 720x g for 8 min at 4°C. The supernatant was removed and the pellet was resuspended in 10 ml GBSS-B solution containing 150 µl DNAse I stock solution and the falcon was filled up to 50 ml with GBSS-B solution. In the next step, the solution was centrifuged at 720x g for 8 min at 4°C and the supernatant was removed. Next, the pellet was resuspended in 10 ml GBSS-B containing 150 µl DNAse I stock solution. In addition, 24 ml GBSS-B and 14 ml of the Nycodenz® 1 solution was added and the solution was carefully mixed. Further, 4 ml of the Nycodenz® 2 solution was transferred into six 15 ml falcon tubes. Next, 8.3 ml of the mixed cell solution containing GBSS-B, DNAse and Nycodenz® 1 was carefully laid onto the Nycodenz® 2 solution and the GBSS-B solution was used to gently overlay the cell suspension to obtain a final volume of 15 ml. Consequently, the gradient solution was centrifuged without brakes at 3000x g for 20 min at 4°C. After the centrifugation step, HSCs are found in the upper gradient phase as a white ring and KCs/LECs are found in the lower gradient phase. Each cell type was carefully transferred to a new falcon and washed with GBSS-B solution and subsequently centrifuged at 720x g for 8 min at 4°C to pellet the cells. The supernatant was removed and subsequently frozen in liquid nitrogen and stored at -80°C for further RNA isolation.

Liver leucocyte isolation and flow cytometry
Livers were dissected, cut into small segments, and digested with 0.5mg/ml of Liberase TM (Roche) in RPMI 1640 (Gibco) for 40 minutes, with agitation at 37 o C. The digested tissue was put on ice and 100µl of 0.1M EDTA was added to stop digestion, followed by passing through a 40-µm mesh cell strainer (Corning) and centrifugation for 6 min at 170g to remove hepatocytes.
Supernatant was collected, centrifuge at 300g for 10 minutes, and the pellets treated with ACK lysis buffer (Thermo Fisher Scientific) to remove red blood cells. After washing, cells were resuspended in RPMI 1640, passed again through a cell strainer, centrifuged, and resuspended in RPMI. Cell counting was performed using a Neubauer chamber after staining with trypan blue to exclude dead cells. For flow cytometry analysis, cells were resuspended in FACS buffer (PBS containing 5% bovine serum albumin and 0.1% sodium azide), followed by Fc receptor blocking with anti-CD16/CD32 antibodies (BD Biosciences). Cells were then stained with fluorochrome-conjugated antibodies to CD45, CD3, TCRβ, TCRδ, CD4, CD8, NK1.1 and CD19; all from BD Biosciences. Data were acquired with a BD FACSCalibur flow cytometer and analyzed using FlowJo™ v10 Software (BD Biosciences).

Quantitative real-time PCR (qPCR)
Trizol reagent (Invitrogen, Karlsruhe, Germany) was used to isolate total RNA from liver tissues. Reverse-transcription was performed using an Omniscript RT Kit (Qiagen Iberia SL, Barcelona, Spain)) according to the instructions.
Relative quantitative gene expression was measured via real-time PCR using a 7900HT Fast Real Time PCR System with SDS software 2.3 (Applied Biosystems, Foster City, CA) and a SYBR Green PCR Kit (Invitrogen).
GAPDH expression was used as internal standard. Primers are available upon request.

Histological evaluation and immunofluorescence staining
Hepatic tissue was fixed in 4% paraformaldehyde (PFA) immediately after extraction, embedded in paraffin, sectioned and stained for H&E or Sirius red.
Samples were reviewed by a pathologist blinded to and who analyzed the degree of liver injury. The percentage of Sirius red (SR) area fraction in all animals was quantified on 10 low-power (magnification, X10) fields per slide, using NIH ImageJ® software (http://rsbweb.nih.gov/). Immunohistochemistry for 4-HNE (Abcam, Cambridge, UK) on paraffin sections was performed.
Briefly, liver sections were deparaffinized with xylene and rehydrated with serially descending percentages of ethanol. The sections were then boiled in 10 mM sodium citrate acid buffer (pH: 6,0) in order to retrieve the antigen followed by incubation with 0.3% Tween20. Afterwards, sections were